The mTOR pathway is a signaling network that controls growth (mass accumulation) and metabolism in response to the nutritional state of organisms. The pathway is commonly deregulated in cancer, neurological disorders like epilepsy, and diabetes, and also modulates the aging process. Indeed, pharmacological or genetic inhibition of mTOR is amongst the best validated approaches for increasing the lifespan of animals. The mTOR protein kinase is the target of the drug rapamycin and the catalytic subunit of two multi-protein complexes, mTOR Complex 1 (mTORC1) and 2 (mTORC2), that nucleate distinct branches of the pathway and respond to different stimuli. mTORC1 responds to a variety of signals, including diverse types of growth factors, nutrients, and stresses, and regulates many anabolic and catabolic processes, including protein, nucleotide, and lipid synthesis as well as autophagy, respectively. Recently, we discovered that mTORC1 senses nutrients in two compartments, the lysosome and the cytosol, and uncovered many of the molecular components involved. While we have made progress in understanding the functions of many of these components, one in particular, GATOR2, has been frustratingly mysterious. We know this protein complex is very important as it binds several nutrient sensors and its loss inhibits mTORC1 activity. However, we still do not understand its biochemical function or structure or function in vivo. We have recently made progress on all of these fronts and now have assays to detect its activity and approaches to isolate its regulation by the amino acid leucine in vivo. The goals of our proposed work are to understand the function and structure of GATOR2 (Aim 1); the role of the GATOR2-interacting leucine sensor Sestrin2 in the adaptation of mice to a leucine-free diet (Aim 2); and the mechanism through which mTORC1 senses glucose in AMPK-independent fashion (Aim 3). We will accomplish these goals with a multi-disciplinary approach that uses the tools of biochemistry, structural and molecular biology, and mouse engineering and analyses. Our results will substantially increase our understanding of a central growth regulator and reveal the function of a component (GATOR2) that may be of value to target in certain disease states.